Protein Synthesis

From Gene to
Protein
Unit 3
Protein synthesis
 The information content of DNA
 Is in the form of specific sequences of nucleotides along
the DNA strands
 The DNA inherited by an organism
 Leads to specific traits by dictating the synthesis of

proteins
 The process by which DNA directs protein synthesis, gene
expression
 Includes two stages, called transcription and translation
 The ribosome
 Is part of the cellular machinery for translation,
polypeptide synthesis
 Genes specify proteins via transcription and translation
 Transcription involves the transfer of genetic information from DNA into an
RNA molecule while translation involves the transfer of the information in the
RNA to the synthesis of a protein
Evidence from the Study of Metabolic
Defects
 The relationship between genes and proteins was first proposed in 1909 by an English physician Archibald Garrod
 He was the first to suggest that genes dictate phenotypes through enzymes which are proteins that catalyze
specific chemical reactions in the cell.
 He hypothesized that inherited diseases reflect a person’s inability to make a particular enzyme.
 Citing the disease alkaptonuria where urine appears dark red due to the presence of alkapton as an example,
Garrod reasoned that normal individuals have an enzyme that breaks down alkapton while alkaptonuric
individuals lack the enzyme
 Garrod’s hypothesis was ahead of its time but research decades later proved him right
Nutritional Mutants in Neurospora:
Scientific Inquiry
 In 1940s, George Beadle and Edward Tatum proved the relationship between genes
and enzymes by using the bread mold, Neurospora crassa.
 Beadle and Tatum studied strains of the mold that were unable to grow on the usual
minimal growth medium. These strains were mutants created using X-ray radiation.
 Each of these mutants lacked an enzyme in a metabolic pathway and therefore were
unable to produce a particular molecule such as an amino acid.
 They showed that each mutant was defective in a single gene and hypothesized that
one gene controlled the production of one specific enzyme.
 This hypothesis has now been modified from one gene-one enzyme to one gene-one
protein to one gene–one polypeptide.
 Using genetic crosses
 Tatum and Beadle determined that their mutants fell into

three classes, each mutated in a different gene

Working with the mold Neurospora crassa, George Beadle and Edward Tatum had isolated mutants requiring
EXPERIMENT arginine in their growth medium and had shown genetically that these mutants fell into three classes, each
defective in a different gene. From other considerations, they suspected that the metabolic pathway of arginine
biosynthesis included the precursors ornithine and citrulline. Their most famous experiment, shown here,
tested both their one gene–one enzyme hypothesis and their postulated arginine pathway. In this experiment,
they grew their three classes of mutants under the four different conditions shown in the Results section below.

RESULTS The wild-type strain required only the minimal medium for growth. The three classes of mutants had
different growth requirements

Class I Class II Class III

Wild type Mutants Mutants Mutants
Minimal
medium
(MM)
(control)
MM +
Ornithine

MM +
Citrulline

MM +
Arginine
(control)
CONCLUSION From the growth patterns of the mutants, Beadle and Tatum deduced that each mutant was unable
to carry out one step in the pathway for synthesizing arginine, presumably because it lacked the
necessary enzyme. Because each of their mutants was mutated in a single gene, they concluded that
each mutated gene must normally dictate the production of one enzyme. Their results supported the
one gene–one enzyme hypothesis and also confirmed the arginine pathway.
(Notice that a mutant can grow only if supplied with a compound made after the defective step.)

Class I Class II Class III

Mutants Mutants Mutants
(mutation (mutation (mutation
Wild type in gene A) in gene B) in gene C)

Precursor Precursor Precursor Precursor

Enzyme
Gene A A A A A

Ornithine Ornithine Ornithine Ornithine

Enzyme
Gene B B B B B
Citrulline Citrulline Citrulline Citrulline

Enzyme
Gene C C C C C
Arginine Arginine Arginine Arginine
The Products of Gene Expression: A
Developing Story
 Beadle and Tatum developed the “one gene–one enzyme hypothesis”
 Which states that the function of a gene is to dictate the production of a specific
enzyme
 As researchers learned more about proteins
 They made minor revision to the one gene–one enzyme hypothesis
 Genes are now known to code for polypeptide chains or for RNA molecules.
 Basic Principles of Transcription
and Translation
 Transcription
 Is the synthesis of RNA under the direction of DNA

 Produces messenger RNA (mRNA)

 Translation
 Is the actual synthesis of a polypeptide, which

occurs under the direction of mRNA

 Occurs on ribosomes
 In prokaryotes
 Transcription and translation occur together

DNA
TRANSCRIPTION

mRNA
Ribosome
TRANSLATION

Polypeptide

(a) Prokaryotic cell. In a cell lacking a nucleus, mRNA

produced by transcription is immediately translated
without additional processing.
 In eukaryotes
 RNA transcripts are modified before becoming true
mRNA

Nuclear
envelope

TRANSCRIPTION DNA

Pre-mRNA
RNA PROCESSING

mRNA

Ribosome

TRANSLATION
(b) Eukaryotic cell. The nucleus provides a separate
Polypeptide compartment for transcription. The original RNA
transcript, called pre-mRNA, is processed in various
ways before leaving the nucleus as mRNA.
 Cells are governed by a cellular chain of
command
 DNA → RNA → protein
The Genetic Code
 How many bases correspond to an amino acid?
 A sequence of three bases known as a base triplet or a codon encode for one amino acid.
 Genetic information
 Is encoded as a sequence of non-overlapping base triplets, or codons
 During transcription
 The gene determines the sequence of bases

along the length of an mRNA molecule

DNA Gene 2
molecule
Gene 1
Gene 3

DNA strand 3′ 5′
(template) A C C A A A C C G A G T

TRANSCRIPTION

U G G U U U G G C U C A
mRNA 5′ 3′
Codon
TRANSLATION

Protein Trp Phe Gly Ser

Amino acid
The Dictionary of the genetic code
Second mRNA base
U C A G
UUU UCU UAU UGU U
Phe Tyr Cys
UUC UCC UAC UGC C
U
UUA UCA Ser UAA Stop UGA Stop A
UUG Leu UCG UAG Stop

Third mRNA base (3′ end)

UGG Trp G

First mRNA base (5′ end)

 A codon in
CUU CCU CAU CGU U
messenger RNA CUC CCC CAC
His
CGC C
C Leu CCA Pro Arg
 Is either CUA CAA CGA A
Gln
CUG CCG CAG CGG G
translated
AUU ACU AAU AGU U
into an amino AUC lle ACC AAC
Asn
AGC Ser C
A Thr
acid or serves AUA ACA AAA
Lys
AGA A
AGG Arg G
Met or
AUG ACG AAG
as a start
GUU GCU GAU GGU U
translational Asp C
G GUC Val
GCC
Ala
GAC GGC
Gly
stop signal GUA GCA GAA GGA A
GUG GCG GAG Glu GGG G
 Codons must be read in the correct reading frame from the 5’ end to the 3’
end without over-lapping
 For the specified polypeptide to be produced
 There are 64 codons out of which 61 codons encode for amino
acids while the remaining 3 act as stop codons to terminate
transcription and translation.
 The stop codons are UAA, UGA, UAG
 AUG is the start codon which also encodes for the amino acid
Methionine.
 The genetic code has redundancy whereby one amino acid can be
encoded for by more than one codon. The maximum number of
codons is 6 while the minimum is 1.
Evolution of the Genetic Code
 The genetic code is nearly universal
 Shared by organisms from the simplest bacteria to the most complex animals
 In laboratory experiments
 Genes can be transcribed and translated after being transplanted from one
species to another
Transcription
 Transcription is the DNA-directed synthesis
of mRNA: a closer look
 Molecular Components of
Transcription
 mRNA synthesis
 Is catalyzed by RNA polymerase, which pries the
DNA strands apart and hooks together the RNA
nucleotides
 Follows the same base-pairing rules as DNA,
except that in RNA, uracil substitutes for thymine
Synthesis of an mRNA Transcript Promoter
Transcription unit
5′ 3′
3′ 5′
DNA
Start point
RNA polymerase 1 Initiation. After RNA polymerase binds to
the promoter, the DNA strands unwind, and
the polymerase initiates RNA synthesis at the
 The stages of start point on the template strand.

transcription are
5′ 3′
3′ 5′
Template strand of
Unwound RNA DNA
 Initiation DNA transcript
2 Elongation. The polymerase moves downstream, unwinding the
 Elongation DNA and elongating the RNA transcript 5′ → 3 ′ . In the wake of
transcription, the DNA strands re-form a double helix.
Rewound
 Termination RNA
5′ 3′
3′ 3′ 5′
5′

RNA
transcript
3 Termination. Eventually, the RNA
transcript is released, and the
polymerase detaches from the DNA.
5′ 3′
3′ 5′

5′ 3′
Completed RNA
transcript
 Elongation Non-template
strand of DNA
RNA nucleotides

RNA
polymerase

T C C A A
A T
3′ C T U
3′ end
T

G
A U

G
G
A
C A U G C A C
5′ A
T A
A G G T T

Direction of transcription
5′ Template
(“downstream”)
strand of DNA

Newly made
RNA
Elongation of the RNA Strand
 As RNA polymerase moves along the DNA
 It continues to untwist the double helix, exposing
about 10 to 20 DNA bases at a time for pairing
with RNA nucleotides
Termination of Transcription
 The mechanisms of termination
 Are different in prokaryotes and eukaryotes
 RNA Processing/Post-Transcriptional
Modification
 Eukaryotic cells modify RNA after
transcription
 Enzymes in the eukaryotic nucleus
 Modify pre-mRNA in specific ways before the
genetic messages are dispatched to the cytoplasm
 They modify the 5’ and 3’ ends and also remove the
introns to splice the exons together to form a
continuous reading frame.
 Alteration of mRNA Ends
 Each end of a pre-mRNA molecule is
modified in a particular way
 The 5′ end receives a modified nucleotide cap
 The 3′ end gets a poly-A tail
A modified guanine nucleotide 50 to 250 adenine nucleotides
added to the 5′ end added to the 3′ end
TRANSCRIPTION DNA

RNA PROCESSING Pre-mRNA Protein-coding segment Polyadenylation signal

5′ 3′
mRNA
G P P P AAUAAA AAA…AAA
Ribosome
TRANSLATION
Start codon Stop codon
5′ Cap 5′ UTR 3′ UTR Poly-A tail
Polypeptide
RNA Splicing
 Removes introns and joins exons to produce a
continuous reading frame
5′ Exon Intron Exon Intron Exon 3′
TRANSCRIPTION DNA Pre-mRNA 5′ Cap Poly-A tail
1 30 31 104 105 146
RNA PROCESSING Pre-mRNA

mRNA Coding Introns cut out and

Ribosome segment exons spliced together
TRANSLATION

Polypeptide
mRNA 5′ Cap Poly-A tail
1 146
3′ UTR 3′ UTR
 Is carried out by spliceosomes in some cases

RNA transcript (pre-mRNA)

5′
Exon 1 Intron Exon 2

Protein
1
snRNA Other proteins

snRNPs
Spliceosome

2 5′

Spliceosome
components
Cut-out
intron
3
mRNA
5′
Exon 1 Exon 2
Translation
 Translation is the RNA-directed synthesis
of a polypeptide: a closer look
Molecular Components of
Translation
 A cell translates an mRNA message into
protein
 With the help of transfer RNA (tRNA)
 Translation: the basic concept
TRANSCRIPTION DNA

mRNA
Ribosome
TRANSLATION
Polypeptide

Amino
Polypeptide acids

tRNA with
amino acid
Ribosome attached
Trp
Ph e Gly

tRNA
C
C
GC G
A
Anticodon
A A A
U G G U U U G G C

5′ Codons 3′
mRNA
 Molecules of tRNA are not all identical
 Each carries a specific amino acid on one end (3’
end)
 Each has an anticodon on the other end which is
complementary to a codon of mRNA
 The Structure and Function of
Transfer RNA
A
C
 A tRNA molecule C

 Consists of a single 3′
RNA strand that is only Amino acid A
C
attachment site C
about 80 nucleotides A 5′
C G
long G C
C G
U G
 Is roughly L-shaped U A
A U
U C A U
* C A C AG UA A G *
G * C U C
*
C G U G U * C G A G G
* * U C * A G G
* G AG C
(a) Two-dimensional structure. The four base-paired regions and three G C Hydrogen
loops are characteristic of all tRNAs, as is the base sequence of the U A bonds
amino acid attachment site at the 3′ end. The anticodon triplet is * G
A
unique to each tRNA type. (The asterisks mark bases that have been A* C
chemically modified, a characteristic of tRNA.) * U
A G
A
Anticodon
 Amino acid
5′
attachment site
3′

Hydrogen
bonds

A AG

3′ 5′
Anticodon
Anticodon
(c) Symbol used
(b) Three-dimensional structure in this book
 A specific enzyme called an aminoacyl-
tRNA synthetase
 Joins each amino acid to the correct tRNA

Amino acid Aminoacyl-tRNA

synthetase (enzyme)

P P P Adenosine
1 Active site binds the
amino acid and ATP.
ATP

2 ATP loses two P groups

and joins amino acid as AMP.
P Adenosine

Pyrophosphate P Pi

Pi
Pi
Phosphates
tRNA
3 Appropriate
tRNA covalently
Bonds to amino
Acid, displacing P Adenosine
AMP. AMP
4 Activated amino acid
is released by the enzyme.

Aminoacyl tRNA
(an “activated
amino acid”)
Ribosomes
 Facilitate the specific coupling of tRNA
anticodons with mRNA codons during protein
synthesis
 The ribosomal subunits
 Are constructed of proteins and RNA
molecules named ribosomal RNA or rRNA

TRANSCRIPTION DNA

mRNA
Ribosome
TRANSLATION

Polypeptide
Exit tunnel
Growing
polypeptide
tRNA
molecules
Large
subunit
E
P A

Small
subunit

5′
mRNA 3′

(a) Computer model of functioning ribosome. This is a model of a bacterial

ribosome, showing its overall shape. The eukaryotic ribosome is roughly
similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules
and proteins.
 P site (Peptidyl-tRNA
binding site)
 The ribosome A site (Aminoacyl-
tRNA binding site)
has three binding E site
sites for tRNA (Exit site)

 The P site Large

subunit
 The A site
E P A
 The E site
mRNA
binding site Small
subunit

(b) Schematic model showing binding sites. A ribosome has an mRNA

binding site and three tRNA binding sites, known as the A, P, and E
sites. This schematic ribosome will appear in later diagrams.
 Amino end Growing polypeptide

Next amino acid

to be added to
polypeptide chain

tRNA

mRNA 3′

Codons
5′

(c) Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon
base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing
polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the
polypeptide chain. Discharged tRNA leaves via the E site.
Building a Polypeptide
 We can divide translation into three stages
 Initiation
 Elongation
 Termination
Initiation
 The initiation stage of translation
 Brings together mRNA, tRNA bearing the first amino

acid of the polypeptide, and two subunits of a ribosome

Large
ribosomal
P site subunit
3′ U A C 5′
t t
Me 5′ A U G3′ Me

Initiator tRNA
GTP GDP
E A
mRNA
5′ 3′ 5′ 3′
Start codon

mRNA binding site Small Translation initiation complex

ribosomal
subunit
1 A small ribosomal subunit binds to a molecule of
mRNA. In a prokaryotic cell, the mRNA binding site
on this subunit recognizes a specific nucleotide
sequence on the mRNA just upstream of the start
codon. An initiator tRNA, with the anticodon UAC,
base-pairs with the start codon, AUG. This tRNA
carries the amino acid methionine (Met).
2 The arrival of a large ribosomal subunit completes
the initiation complex. Proteins called initiation
factors (not shown) are required to bring all the
translation components together. GTP provides
the energy for the assembly. The initiator tRNA is
in the P site; the A site is available to the tRNA
bearing the next amino acid.
Elongation
 In the elongation stage of translation
 Amino acids are added one by one to the preceding amino
acid
1 Codon recognition. The anticodon
TRANSCRIPTION DNA
Amino end of an incoming aminoacyl tRNA
mRNA
of polypeptide base-pairs with the complementary
Ribosome
TRANSLATION mRNA codon in the A site. Hydrolysis
Polypeptide
of GTP increases the accuracy and
E efficiency of this step.
mRNA 3′
Ribosome ready for P A
5′ site site
next aminoacyl tRNA
2 GTP
2 GDP

E E

P A P A

2 Peptide bond formation. An

GDP rRNA molecule of the large
3 Translocation. The ribosome GTP subunit catalyzes the formation
translocates the tRNA in the A
of a peptide bond between the
site to the P site. The empty tRNA
new amino acid in the A site and
in the P site is moved to the E site, E
the carboxyl end of the growing
where it is released. The mRNA
polypeptide in the P site. This step
moves along with its bound tRNAs, P A attaches the polypeptide to the
bringing the next codon to be
tRNA in the A site.
translated into the A site.
Termination
 The final stage of translation is termination
 When the ribosome reaches a stop codon in
the mRNA
Release
factor
Free
polypeptide

5′
3′ 3′
3′
5′ 5′
Stop codon
(UAG, UAA, or UGA)
1 When a ribosome reaches a stop 2 The release factor hydrolyzes 3 The two ribosomal subunits
codon on mRNA, the A site of the the bond between the tRNA in and the other components of
ribosome accepts a protein called the P site and the last amino the assembly dissociate.
a release factor instead of tRNA. acid of the polypeptide chain.
The polypeptide is thus freed
from the ribosome.
Polyribosomes
 A number of ribosomes can translate a single mRNA molecule
simultaneously
 Forming a polyribosome

Completed
Growing polypeptide
polypeptides
Incoming
ribosomal
subunits
Start of Polyribosom
e End of
mRNA mRNA
(5′ end) (3′ end)
(a) An mRNA molecule is generally translated simultaneously
by several ribosomes in clusters called polyribosomes.

Ribosomes
mRNA

0.1 µm
(b) This micrograph shows a large polyribosome in a prokaryotic
cell (TEM).
Completing and Targeting the
Functional Protein
 Polypeptide chains
 Undergo modifications after the translation
process
Protein Folding and Post-
Translational Modifications
 After translation
 Proteins may be modified in ways that affect their
three-dimensional shape
Targeting Polypeptides to Specific
Locations
 Two populations of ribosomes are evident
in cells
 Free and bound
 Free ribosomes in the cytosol
 Initiate the synthesis of all proteins
 Proteins destined for the endomembrane
system or for secretion
 Must be transported into the ER
 Have signal peptides to which a signal-
recognition particle (SRP) binds, enabling the
translation ribosome to bind to the ER
  The signal mechanism for targeting
proteins to the ER
1 Polypeptide 2 An SRP binds 3 The SRP binds to a 4 The SRP leaves, and 5 The signal- 6 The rest of
synthesis begins to the signal receptor protein in the ER the polypeptide resumes cleaving the completed
on a free peptide, halting membrane. This receptor growing, meanwhile enzyme polypeptide leaves
ribosome in synthesis is part of a protein complex translocating across the cuts off the the ribosome and
the cytosol. momentarily. (a translocation complex) membrane. (The signal signal peptide. folds into its final
that has a membrane pore peptide stays attached conformation.
and a signal-cleaving enzyme. to the membrane.)

Ribosome

mRNA
Signal
peptide ER
membrane
Signal
Signal-
peptide
recognition Protein
removed
particle
(SRP) SRP
receptor
CYTOSOL protein

Translocation
ERLUMEN
complex
 Comparing gene expression in prokaryotes and
eukaryotes reveals key differences
 Prokaryotic cells lack a nuclear envelope
 Allowing translation to begin while transcription is still

in progress

RNA polymerase

DNA
mRNA
Polyribosome
Direction of 0.25 µ m
RNA
transcription
polymerase
DNA

Polyribosome
Polypeptide
(amino end)
Ribosome
mRNA (5′ end)
 In a eukaryotic cell
 The nuclear envelope separates transcription
from translation
 Extensive RNA processing occurs in the nucleus
 Point Mutations

 Point mutations can affect protein structure

and function
 Mutations
 Are changes in the genetic material of a cell
 Point mutations
 Are changes in just one base pair of a gene
  The change of a single nucleotide in the
DNA’s template strand
 Leads to the production of an abnormal protein

Wild-type hemoglobin DNA Mutant hemoglobin DNA In the DNA, the

3′ 5′ 3′ 5′ mutant template
C T T C A T strand has an A where
the wild-type template
has a T.

mRNA mRNA
The mutant mRNA has
G A A G U A a U instead of an A in
one codon.
5′ 3′ 5′ 3′

Normal hemoglobin Sickle-cell hemoglobin

The mutant (sickle-cell)
Glu Val hemoglobin has a valine
(Val) instead of a glutamic
acid (Glu).
Types of Point Mutations
 Point mutations within a gene can be
divided into two general categories
 Base-pair substitutions
 Base-pair insertions or deletions
Substitutions
Wild type

 A base-pair substitution mRNA

Protein
5′
A U G
Met
A A G U U U GG C U A A
Lys Phe Gly
3′
Stop
 Is the replacement of Amino end
Base-pair substitution
Carboxyl end

one nucleotide and its No effect on amino acid sequence

U instead of C

partner with another A U G A A G U U U G G U U A A

pair of nucleotides Missense

Met Lys Phe Gly
A instead of G
Stop

 Can cause mis-sense A U G A A G U U U A G U U A A

or nonsense Met Lys Phe Ser Stop

Nonsense
U instead of A
A U G U A G U U U G G C U A A
Met Stop
Insertions and Deletions
Wild type
 Insertions and deletions mRNA 5′
A UG A A GU U U GG C U A A
3′
Protein Met Lys Phe Gly Stop
 Are additions or losses Amino end Carboxyl end
Base-pair insertion or deletion
of nucleotide pairs in a Frameshift causing immediate nonsense
Extra U
gene AU G U A AG U U U G GC U A

 May produce Met

Frameshift causing
Stop

frameshift mutations extensive missense U Missing

A U G A A GU U G G C U A A
Met Lys Leu Ala
Insertion or deletion of 3 nucleotides:
no frameshift but extra or missing amino acid
A AG Missing

A U G U U U G G C U A A
Met Phe Gly
Stop
Mutagens
 Spontaneous mutations
 Can occur during DNA replication,
recombination, or repair
 Mutagens
 Are physical or chemical agents that can cause
mutations
What is a gene? revisiting the
question
 A gene
 Is a region of DNA whose final product is either a
polypeptide or an RNA molecule
 A summary of transcription and translation in
a eukaryotic cell
TRANSCRIPTION DNA
1 RNA is transcribed
from a DNA template.
3′
A
ly-
Po

5′ RNA RNA
transcript polymerase
RNA PROCESSING Exon
2 In eukaryotes, the
RNA transcript
RNA transcript (pre-
(pre-mRNA)
mRNA) is spliced and
Intron
modified to produce
mRNA, which moves Aminoacyl-tRNA
p synthetase
from the nucleus to the Ca
cytoplasm. NUCLEUS

Amino
FORMATION OF
acid
INITIATION COMPLEX AMINO ACID ACTIVATION
CYTOPLASM 3 After leaving the tRNA
4 Each amino acid
nucleus, mRNA attaches attaches to its proper tRNA
to the ribosome. with the help of a specific
enzyme and ATP.
mRNA Growing
polypeptide
A Activated
ly -
Po amino acid
A
ly -
Ribosomal Po
subunits

p
Ca
5′
TRANSLATION
C 5 A succession of tRNAs
AC U
E A AC add their amino acids to
the polypeptide chain
AAA Anticodon
as the mRNA is moved
UG GUU UA U G through the ribosome
one codon at a time.
Codon (When completed, the
polypeptide is released
Ribosome from the ribosome.)
Try this!
 1. What are transcription and translation?
 2. How many nucleotides are necessary to code for a polypeptide that is
100 amino acids long?
 3. An mRNA molecule contains the nucleotide sequence
CCAUUUACG. Using the dictionary of the genetic code, translate this
sequence into the corresponding amino acid sequence.
 4. What is an anticodon?
 5. What is the function of the ribosome in protein synthesis?
 6. Which of the following does not participate directly in translation:
ribosomes, tRNA, mRNA, DNA, enzymes and ATP?